Multicopper oxidases (MCOs) are a family of enzymes that include laccases (p-diphenol: dioxygen oxidoreductases, EC 1.10.3.2), ascorbate oxidases (EC 1.10.3.3), ferroxidases (EC 1.16.3.1), bilirubin oxidases (EC 1.3.3.5) and other enzyme subfamilies (Solomon et al, 1996; Hoegger et al., 2006). MCOs couple the oxidation of organic and/or inorganic substrates to the four-electron reduction of molecular oxygen to water. These enzymes often have four Cu atoms classified into Type 1 (T1), Type 2 (T2) and Type 3 (T3) centers, in which a mononuclear T1 center on the surface of the enzyme provides long range intramolecular one-electron transfer from electron-donating substrates to an internal trinuclear T2/T3 center formed by a T2 Cu coordinated to a T3 Cu pair. The T2/T3 cluster subsequently reduces dioxygen to water.
Enzymes of the laccase subfamily oxidize a broad range of compounds including phenols, polyphenols, aromatic amines, and non-phenolic substrates by one-electron transfer to molecular oxygen and, thus, have a wide variety of applications spanning from biofuels to human health. A lacquer tree laccase (from Rhus vernicifera) has been used in paint and adhesives in East Asia for more than 6000 years (Hüttermann et al., 2001). Laccases have also been used in the delignification of pulp, bleaching of textile and carcinogenic dyes, detoxification of water and soils, removal of phenolics from wines, improving adhesive properties of lignocellulosic products, determination of bilirubin levels in serum, and transformation of antibiotics and steroids (Sakurai et al., 2007). Likewise, laccases have demonstrated potential in biosensors, bioreactors and biofuel cells (Shleev et al., 2005).
Although laccases were once thought to be restricted to eukaryotes (fungi, plants, insects), recent evidence suggests their widespread distribution in bacteria (Claus, 2004). In plants, laccases are required for normal cell wall structure and integrity in xylem fibers and apparently involved in lignification (Ranocha et al., 2002). In fungi, laccases mediate the modification and degradation of complex natural polymers such as lignin and humic acids (Widsten et al, 2008; Claus et al., 1998). The lacasse-like MCOs of insects seem to play an important role in cuticular sclerotization, melanization, iron homeostasis and the oxidation of toxic compounds in the diet (Claus, 2004). Likewise, the more recently described laccase-like MCOs of bacteria have a wide variety of biological roles including sporulation, electron transport, pigmentation, metal (copper, iron, manganese) homeostasis, oxidation of phenolate-siderophorcs, phenoxazinone synthesis, cell division and morphorgenesis (Claus, 2003). For example, Martins et al. (2002) characterized a laccase enzyme isolated from the endospore coat of Bacillus subtilis. Koschorreck et al. (2008) recently disclosed a laccase isolated from Bacillus licheniformis and found it to catalyze dimerization of phenolic acids.
However, these laccases usually have very limited temperature, pH, and salt range because of the living conditions of the plants and bacteria. Their industrial application, which sometimes requires high laccase activity under extreme conditions, may, thus, be further limited. Therefore, laccases with extreme thermal and salt/solvent stability are highly desired.
Archaea, one of the three domains of life along with the Bacteria and Eukarya, have evolved to thrive in harsh environmental conditions including high temperature, extreme pH, and/or low water activity. Thus, their systems and enzymes are deemed ideal for a number of industrial processes. In contrast to the widespread occurrence of laccases in eukaryotes and bacteria, only a few putative MCOs are predicted in genomes of archaea including the hyperthermophilic crenarchaeote Pyrobaculum aerophilum (PAE1888) (Fitz-Gibbon et al., 2002) and the PAE1888-encoded enzyme is not likely to catalyze the oxidation of phenolic compounds particularly in the absence of metal supplementation. This limitation in PAE1888 activity is based on the inventors' dendrogram analysis (see report) and transcriptional mapping by Cozen et al. (2009, J. Bacteriol. 191(3): 782-794), both of which suggest PAE1888 is not a true laccase and instead encodes a metal oxidase and/or NO2− or N2O reductase. In addition, whereas all of the archaea with identified MCO grow in the presence of oxygen, most archaea with sequenced genomes are strict anaerobes, likely limiting the distribution of the oxygen-utilizing MCOs among this group.